The polo subfamily of protein kinases has been identified in various eucaryotic organisms and appears to play pivotal roles in cell division and proliferation. The polo subfamily members that function in mitosis and cytokinesis include mammalian Plk, Xenopus laevis Plx1, Drosophila melanogaster polo, Schizosaccharomyces pombe Plo1, and Saccharomyces cerevisiae Cdc5. A feature of the polo subfamily members is the presence of a distinct region of homology in the C-terminal non- catalytic domain, termed the polo-box. Mammalian polo-like kinase, Plk, is a functional homologue of S. cerevisiae Cdc5. In wild-type yeast, ectopic expression of Plk can induce abnormally elongated buds with additional septal structures. Plk localizes at the spindle poles, cytokinetic neck filaments, and ectopic septal structures. Without impairing kinase activity, mutations in the polo-box disrupt the capacity of Plk to complement the defect associated with a cdc5-1 temperature-sensitive mutation, to induce elongated buds, and to localize to the subcellular structures. Likewise, Cdc5 also localizes at the sites where Plk localizes, and mutations in the polo-box disrupt both its localization and function. Consistent with these results, the C-terminal domain of Plk/Cdc5, but not its polo-box mutant, is sufficient for subcellular localization, and overexpression of the polo-box domain of Cdc5, but not a mutated polo-box, resulted in chains of connected cells. These cells share cytoplasm within the connected cell bodies and have normal nuclear division, indicating that cytokinesis is inhibited without affecting the cell division cycle. Since introduction of multiple copies of Cdc5 partially reverts this defect, it is apparently due to a dominant-negative effect of the kinase domain-deficient polo-box region, disrupting the interaction between endogenous Cdc5 and its binding partner(s) essential for cytokinesis. Our data provide evidence that the polo-box plays a critical role for the function of polo kinases in cytokinesis. - Cell Cycle, mitosis, cytokinesis, Plk, Cdc5,